.alpha.v.beta.3 is a member of the integrin supergene family of cell-surface glycoprotein receptors that promote cellular adhesion. Each cell has a specific repertoire of receptors that define its adhesive capabilities. The integrins are expressed as heterodimers of noncovalently associated .alpha. and .beta. subunits. According to the nomenclature proposed by Hynes, R. O. [Cell 48, 875-886 (1987)], the integrins can be divided into families each with a common .beta.-subunit and a set of variable .alpha.-subunits known to associate with the common .beta.-subunit. The different .alpha. chains are denoted by the original cell type, by a subscript used by the original discoverer, or, as in the case of the .alpha.v.beta.3 receptor, by the nature of the ligand (i.e. .alpha.v stands for a vitronectin receptor .alpha.-chain). Many, but not all, integrin receptors have been shown to interact with proteins via a tripeptide sequence, Arg-Gly-Asp (or RGD using the single letter amino acid code), originally defined from studies of the cell binding domains of fibronectin [Ruoslahti, E. and Pierschbachter, M. D., Cell 44, 5170518 (1986); Ruoslahti, E. and Pierschbachter, M.D., Science 238, 491-497 (1987)].
.alpha.v.beta.3 (also referred to as vitronectin receptor or VNR) is a member of the .beta.3 integrin subfamily and is expressed on a variety of cells, including endothelial, melanoma, smooth muscle cells and, along with another integrin .alpha.2.beta.1 (VLA-2) (the receptor for Type I collagen and laminin), on the surface of osteoclasts [Horton, M. A. and Davies, J., J. Bone Min. Res. 4, 803-808 (1989); Davies, J. et al., J. Cell. Biol. 109, 1817-1826(1989); Horton, M., Int. J. Exp. Pathol. 71 741-759 (1990)]. .alpha.v.beta.3 mediates cell adhesion to vitronectin, fibrinogen, fibronectin, thrombospondin, osteopontin, bone sialo protein II and von Willebrand factor.
Osteoclasts are the main type of bone cells involved in the resorption of bone tissues. The resorption process involves the proliferation and chemotaxis of developing osteoclasts to the skeleton from hematopoietic sites migration of mature cells to sites of subsequent resorption, attachment of osteoclasts to bone substrate and the eventual formation of the polarized, functional mature end cells which are directly involved in bone resorption. The .alpha.v.beta.3 integrin mediates adhesion of osteoclasts to RGD sequence-containing bone matrix proteins.
Antibodies to .alpha.v.beta.3 are expected to be valuable diagnostic and therapeutic tools in studying the biological role and the structural/functional relationships of this integrin with its various ligands. In particular, monoclonal antibodies (Mabs) detecting unique epitopes on osteoclasts would be of great value in understanding of the development of osteoclasts. Even more importantly, neutralizing Mabs specific for .alpha.v.beta.3 that inhibit the osteoclast binding to the bone matrix proteins have great potential as therapeutic agents useful in the treatment of conditions associated with excessive bone resorption.
There are several monoclonal antibodies known in the art that bind to various epitopes on .alpha.v.beta.3. Immunizing with osteoclasts from osteoclastomas (giant cell tumors of bone), Horton, M. A. et al. [Cancer Res. 45, 5663-5669 (1985)] produced eleven mouse hybridomas secreting monoclonal antibodies which bind to osteoclasts in normal human fetal bone and a variety of neoplastic and non-neoplastic bone lesions. One of these, designated 23C6, was subsequently shown to bind the .alpha.v.beta.3 complex, and was demonstrated to be able to disrupt osteoclast function [Horton, M. A. et al., Exp. Cell. Res. 195 368-375 (1991)]. Another monoclonal antibody, LM609 (produced in hybridoma LM609 ATCC HB 9537) disclosed in PCT Application Publication No. WO 89/05155 (published 15 Jun. 1989) and Cheresh et al. J. Biol. Chem. 262:17703-17711 (1987) was also found to bind the .alpha.v.beta.3 complex and, due to its ability to inhibit the binding of ECr molecules present on the surface of tumor cells and blood vessel forming endothelial cells to vitronectin, fibrinogen and von Willebrand factor, was proposed for therapeutic use as tumor growth inhibitor. Monoclonal antibody 13C2 (Horton, M. A. et al., Cancer Res. 1985, Supra) was shown to bind the .alpha.v portion of the .alpha.v.beta.3 molecule, whereas several other monoclonal antibodies were reported to recognize the .beta.3 portion [Nesbitt, S. et al., Epitope Analysis of the Vitronectin Receptor (CD51), In "Leukocyte Typing IV" White Cell Differentiation Antigens, Knapp, W. et al. (eds.) 1991, p. 1037]. The specific monoclonal antibodies variously reported in the art were shown to also bind to endothelial cells and various melanoma cell lines.
There is a need for high affinity monoclonal antibodies to the .alpha.v.beta.3 integrin that are capable of effective inhibition of the binding of .alpha.v.beta.3 expressing cells to .alpha.v.beta.3 ligands, such as vitronectin and fibronectin.
It would be further desirable to provide monoclonal antibodies to .alpha.v.beta.3 that bind osteoclasts and optionally other cells known to express .alpha.v.beta.3.
It would be particularly desirable to provide monoclonal antibodies that are effective inhibitors of .alpha.v.beta.3 binding to its ligands and which specifically bind osteoclasts without binding to other cells known to express .alpha.v.beta.3, i.e., which are more specific for the target integrin on osteoclasts.